Project Summary/Abstract: The broad, long-term objective of this Project is to better understand the underlying mechanisms of immunosuppression following infection in the lungs by examining distinct host- pathogen interplay. Severe acute lower respiratory tract infection or pneumonia remains a major cause of sepsis and risk factor for the acute respiratory distress syndrome (ARDS) worldwide. However, bacterial pneumonia is also a frequent complication of ARDS and prolonged mechanical ventilation that increases patient morbidity, length of stay, and health-care costs. Yet, many aspects of host-pathogen interactions remain poorly understood and the underlying defects in host control mechanisms that occur during critical illness remain outstanding questions in ARDS. We have developed a 2-hit murine model targeting the mononuclear phagocyte system of the liver to study the consequences of severe lung infection induced by Klebsiella pneumoniae, an extracellular Gram-negative pathogen that is a common cause of nosocomial pneumonia, and potential link to systemic immunosuppression. We identified impaired expression of interferon- regulatory factor 1 (IRF-1), a transcription factor critical for boosting antimicrobial and antiviral innate immunity, in this 2-hit model leading us to propose that defects in IRF-1 mediates the immunosuppressive phenotype by impairing transactivation of key innate immunity genes important for host defense. Moreover, preliminary findings suggest that a proportion of critically ill patients exhibit an immunosuppressive phenotype characterized by failure to control relatively avirulent carbapenamase-producing Klebsiella pneumoniae (KP) growth in vitro, and impaired serum killing of KP is associated with defects in alternative but not classical complement activity. Most complement proteins are synthesized in the liver, but macrophages/monocytes also synthesize these factors, many of which are regulated by interferons. Collectively, preliminary findings from the 2-hit model and patient serum have led us to propose a major hypothesis that macrophage dysfunction characterized by defects in IRF-1 marks the host for an immunosuppressive fate during infection due to impaired cytokine response, microbial killing, and complement activity. Aim 1 will determine whether impairment in IRF-1 signaling is a mechanism for defective chemokine and cytokine response in the 2-hit model. Aim 2 will evaluate whether defects in mitochondrial aconitate decarboxylase 1 (ACOD1) downstream of IRF-1 impairs host anti-microbial program during KP infection and in the 2-hit model. Aim 3 will evaluate IRF-1 activation, the dynamics of alternative pathway complement factors synthesized by mononuclear phagocytes, and relationship to AP activity in a cohort of critically ill patients. Successful completion of the aims will elucidate novel mechanisms of host control and aid in the long-term objective of understanding immunosuppressive signals complicating severe pneumonia.